Whole-brain inputs and outputs of Phox2b and GABAergic neurons in the nucleus tractus solitarii

The nucleus tractus solitarii (NTS) plays a critical role in the homeostatic regulation of respiration, blood pressure, sodium consumption and metabolic processes. Despite their significance, the circuitry mechanisms facilitating these diverse physiological functions remain incompletely understood. In this study, we present a whole-brain mapping of both the afferent and efferent connections of Phox2b-expressing and GABAergic neurons within the NTS. Our findings reveal that these neuronal populations not only receive monosynaptic inputs primarily from the medulla oblongata, pons, midbrain, supra-midbrain and cortical areas, but also mutually project their axons to these same locales. Moreover, intense monosynaptic inputs are received from the central amygdala, the paraventricular nucleus of the hypothalamus, the parasubthalamic nucleus and the intermediate reticular nucleus, along with brainstem nuclei explicitly engaged in respiratory regulation. In contrast, both neuronal groups extensively innervate brainstem nuclei associated with respiratory functions, although their projections to regions above the midbrain are comparatively limited. These anatomical findings provide a foundational platform for delineating an anatomical framework essential for dissecting the specific functional mechanisms of these circuits.

To unveil the function-specific neural circuits, it is necessary to determine the afferent and efferent projections of NTS neurons.It is well-established that glutamatergic, catecholaminergic and GABAergic neurons, accounting for the majority of subtypes in the NTS, have been observed to regulate multiple physiological functions.To date, a few studies have demonstrated neuroanatomical connections of NTS neurons with other brain regions.Neural tracing data from Loewy laboratory revealed anatomical and functional connections of the enzyme 11-beta-hydroxysteroid dehydrogenase type 2-expressing NTS neurons (Geerling and Loewy, 2006;Resch et al., 2017;Gasparini et al., 2019).Subsequently, Gasparini and colleagues utilized an axonal tracer (cholera toxin b) to provide a brain-wide map of neurons that extend their axons to the caudal NTS (Gasparini et al., 2020).Recently, we characterized whole-brain monosynaptic inputs and outputs of leptin receptor b-expressing neurons within the NTS (Sun et al., 2023), providing a neuroanatomical basis for further research on physiological and pathological functions of these neurons.NTS Phox2b neurons represent the majority of excitatory neurons, including glutamatergic and catecholaminergic, whereas Phox2b was almost absent in GABAergic NTS (NTS GABA ) neurons (Kang et al., 2007).GABAergic neurons are abundant in the NTS, with a characteristic of both short-and long-range projections (Shi et al., 2021).Nevertheless, studies on connections linking both NTS Phox2b and NTS GABA neurons with other brain regions are required to provide a comprehensive understanding of function-specific neural circuits of these neurons.
In the present studies, we employed transsynaptic retrograde rabies virus and common anterograde virus-based tracing strategies to provide a detailed whole-brain mapping of afferent and efferent projections of both NTS Phox2b and NTS GABA neurons.Building on previous neural tracing evidence, we extended the investigation to concurrently visualize efferent pattern of both excitatory and inhibitory NTS neurons from Phox2b-Flop::Vgat-Cre mice.These data provide a foundational anatomical framework for future investigations into the interregional pathways that regulate NTS functions.

Animal
Adult Phox2b-Cre (IMSR Cat# JAX: 016223), Phox2b-Flpo (IMSR Cat# JAX: 022407) and Vgat-Cre (IMSR Cat# JAX: 016962) mice of either sex used in the experiments were purchased from the Jackson Laboratory.Phox2b-Cre (Scott et al., 2011) and Phox2b-Flpo (Hirsch et al., 2013) mice have Cre or Flpo recombinase expression under control of the Phox2b promoter/ enhancer regions within the BAC transgenic mouse line.Phox2b-Flpo::Vgat-Cre mice were generated by crossing Vgat-Cre mice with Phox2b-Flpo mice.Mice were housed under programcontrolled temperature (23 ± 1°C) and humidity (50% ± 10%) with a fixed 12 h light/12 h dark cycle and with ad libitum access to food and water.All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Ethics Committee of Hebei Medical University.

Surgery and viral injections
Surgical procedures were conducted in accordance with methodologies outlined in prior studies (Sun et al., 2023).Adult mice underwent anesthesia induced by intraperitoneal administration of pentobarbital sodium at a dosage of 60 μg/g.Anesthetic depth was monitored by the absence of corneal and hindpaw withdrawal reflexes, with supplemental anesthetic provided as needed, amounting to 30% of the initial dosage.All procedures were performed under sterile conditions.Post-anesthesia, the mice were positioned prone in a stereotaxic frame (RWD Life Science Co., Ltd., Shenzhen, China) and body temperature was maintained at 37°C using a heating pad and a blanket.Viral vectors were delivered precisely to the NTS at the level of the calamus scriptorius (anterior-posterior (AP): + 0.2 mm, mediallateral (ML): ± 0.3 mm; dorsal-ventral (DV): − 0.2 mm; AP: + 0.3 mm, ML: ± 0.4 mm, DV: − 0.3 mm) using a glass micropipette.For retrograde tracing, injections of helper viruses (100 nL per injection) were administered unilaterally into the NTS and were allowed to diffuse for 10 min.T Following a two-week interval, RV-EnvA-∆G-tdTomato (160 nL) was injected into the same site.Mice were perfused for immunostaining 1 week later.

Histology and immunostaining
Mice were subjected to deep anesthesia using urethane (1.8 g/kg, intraperitoneally) and subsequently perfused transcardially with saline, followed by fixation with 4% paraformaldehyde in phosphatebuffered saline (PBS).Post-fixation, brains were excised on ice and preserved in 4% paraformaldehyde at 4°C for 12 h, after which they were transferred into 30% sucrose solution buffered with PBS for cryoprotection.Coronal sections (25 μm) were harvested on a freezing microtome (CM1950; Leica Microsystems, Germany).For immunohistochemistry, sections were initially blocked using 5% bovine serum albumin in PBS containing 0.25% Triton X-100 for 30 min at room temperature.This step was followed by overnight incubation at 4°C with primary antibodies diluted in PBS supplemented with 2% bovine serum albumin.Sections were subsequently washed thrice in PBS for 5 min each and incubated with fluorescently-labeled secondary antibodies for 2 h at room temperature.All immersion steps and incubations were conducted on a shaker set to a gentle speed.After a final series of washes in PBS, sections were mounted on glass slides using Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, United States) to enhance fluorescence retention and prevent photobleaching during microscopic examination.

Imaging and data analysis
Images of whole-brain sections were acquired using a laser scanning confocal microscope with either 10x or 20x objectives, and detailed magnification was achieved through the employment of a 20x objective on a Zeiss microscope (LSM 900; Carl Zeiss, Jena, Germany).The delimitation of subregional boundaries was referenced against the Mouse Brain Atlas (Paxinos and Franklin, 2013).Quantitative analysis involved the manual enumeration of tdTomato-expressing neurons within distinct nuclei.The relative prevalence of input neurons across 86 brain regions was determined by calculating the ratio of afferent cells in each nucleus to the total population of tdTomato-marked cells, excluding those at the injection sites.Neuronal distributions across each brain region accounted for at least 0.05% of the total monosynaptic inputs were specifically noted.
For the quantification of whole-brain efferent projections emanating from NTS Phox2b and NTS GABA neurons, analyses focused on the proportion of axonal projections, computed as the ratio between the average count of axonal varicosities and the total number of varicosities observed in whole-brain projections.Further, axonal varicosities were documented using a 20x objective on the Zeiss confocal system, and the enumeration of these structures across the brain was performed semi-automatically using a particle analyzing plugin in ImageJ software.Results and statistical analyses were restricted to data derived exclusively from mice with accurately targeted injection sites, as discerned in the accompanying figures.

Quantification and statistical analysis
Statistical analyses were performed using the Prism software version 9 (GraphPad, La Jolla, CA).All data are presented as the mean ± SEM (standard error of the mean).

Experimental strategy to target NTS Phox2b and NTS GABA neurons
To identify the whole-brain monosynaptic inputs onto NTS Phox2b and NTS GABA neurons, we utilized trans-synaptic retrograde labeling with a modified rabies virus as depicted before (Wall et al., 2010;Osakada and Callaway, 2013).This genetic approach entails two stages.Initially, the Cre-dependent helper viruses, including AAV-EF1α-DIO-EGFP-T2A-TVA and AAV-EF1α-DIO-N2cG, were unilaterally injected into the NTS from Phox2b-Cre (n = 3 mice) and Vgat-Cre mice (n = 3 mice), respectively.After 2 weeks, the modified rabies virus RV-EnvA-∆G-tdTomato was administered at the same injection site, targeting selectively infected TVA-expressing cells and transporting retrogradely into presynaptic cells.The mice were perfused 1 week after rabies virus injection, ensuring enough time for the rabies virus to retrogradely infect and express tdTomato sufficiently in the input neurons (Figures 1A,B).Since the rabies virus cannot infect cells in the absence of the TVA receptor, infection is limited to Cre + cells expressing the helper AAV.Starter neurons was identified based on co-expression of AAV helper tag (EGFP) and RV-tdTomato around the injection site, and the input neurons were detected by the expression of tdTomato that directly input to NTS Phox2b and NTS GABA neurons.Through mapping the starter neurons in the NTS across three mouse coronal sections, we observed that they were primarily distributed in rostrocaudal position spanning from bregma −7.6 mm to −7.2 mm.In addition, we identified tdTomato-positive neurons in the NTS that do not express EGFP, indicating the presence of direct monosynaptic inputs to the NTS neurons (Figures 1C-H).However, using the same virus strategy in wild-type mice, we detected no neurons expressing EGFP or tdTomato in the NTS (n = 3, data not shown), demonstrating the reliability of this technique.Here we did not check peripherally monosynaptic inputs to both population of NTS neurons.

Whole-brain input patterns for NTS Phox2b and NTS GABA neurons
To unravel the monosynaptic inputs to NTS Phox2b throughout the entire brain, we examined these input neurons in coronal sections from Phox2b-Cre mice based on the above neural tracing strategy.The neurons exclusively labeled with tdTomato were identified as the monosynaptic inputs to the NTS Phox2b neurons.Examination of representative sections demonstrated that tdTomato-positive neurons were distributed in many brain regions, encompassing the cortex, supra-midbrain, midbrain, pons, medulla oblongata and cerebellum (Figure 2).Using the same strategy in Vgat-Cre mice, tdTomato-labeled neurons were detected in almost the same regions as identified in Phox2b-Cre mice (Figure 3).

Quantitative analysis of input neurons of NTS neurons
To quantify the proportion of monosynaptic input neurons that target both NTS Phox2b and NTS GABA neurons, we made a statistical analysis by calculating the ratio of the number of tdTomato-labeled afferent neurons originating from each nucleus and the total number of labeled neurons in the whole brain (Figure 6, n = 3).The brains were divided into six structures (n = 86 nuclei): cortex, supra-midbrain, midbrain, pons, medulla oblongata and cerebellum.The tdTomatolabeled neurons were counted in each brain region, with each comprising >0.05% of the total labeled neurons.
Collectively, the findings offer a detailed whole-brain mapping of monosynaptic input neurons for both NTS Phox2b and NTS GABA neurons, facilitating the identification of distinct regions implicated in the modulation of various physiological processes for future investigation.

Mapping of efferent projections of both NTS Phox2b and NTS GABA neurons
Next, we characterized the outputs of NTS neurons by mapping their axonal terminals, employing a widely-used anterograde viral tracing strategy as reported in previous studies (Sun et al., 2023).To concurrently visualize the axonal projections of NTS Phox2b and NTS GABA neurons, both AAV-EF1α-DIO-EYFP and AAV-EF1α-fDIO-mCherry were injected into the NTS from Phox2b-Flpo::Vgat-Cre mouse mice (Figure 7A).Four weeks post-injection, immunohistochemical staining revealed that both EYFP-and mCherry-expressing somata were predominantly localized within the NTS.A very small number of fluorescently labeled cells were observed in adjacent regions such as the dorsal nucleus of the vagus nerve (DMNV) and area postrema (AP) (Figure 7B).Here we presented the consistent observations of clear projection fields of both types of NTS neurons across all experiments (n = 3 mice).
To ascertain the projection pattern for both NTS Phox2b and NTS GABA neurons, we quantified pixel of axonal varicosities.Both neuron types exhibited pronounced projections within the medulla oblongata and the pons, while projections were sparser towards the supra-midbrain and midbrain, with minimal to no projections detected reaching the cortex or cerebellum.As illustrated (Figure 9A for NTS Phox2b , Figure 9B for NTS GABA , respectively), statistical analysis revealed that the dense projections from NTS neurons predominantly targeted the IRt (20.28 ± 3.40% for NTS Phox2b , 7.99 ± 0.79% for NTS GABA ).Additionally, the LPBN in the pons (6.42 ± 1.68% for NTS Phox2b , 10.42 ± 0.26% for NTS GABA ) and VLPAG in the midbrain (6.25 ± 2.13% for NTS Phox2b , 6.44 ± 0.89% for NTS GABA ) also received some projections from both types of NTS neurons.In contrast, within the supra-midbrain regions, the BNST (6.41 ± 1.87%) and PSTh (1.66 ± 0.60%) received projections predominantly from NTS Phox2b neurons rather than from NTS GABA neurons, suggesting a differential projection pattern between the two neuron types.

Discussion
In this investigation, we present a comprehensive cerebral atlas that vividly delineates both the afferent and efferent projections of NTS Phox2b and NTS GABA neurons, representing excitatory and inhibitory neuronal types, respectively.Our findings reveal that both distinct neuronal populations not only receive monosynaptic inputs from an expansive range of cerebral territories including the medulla oblongata, pons, midbrain, supra-midbrain, and cortical areas, but also reciprocate by sending axonal outputs to the majority of these identical regions.This detailed mapping accentuates the bidirectional connectivity between the NTS and these significant brain regions.Moreover, the differential projection patterns to specific brain regions by both neuronal types implicate their pivotal roles in distinct function-specific neural circuits.This comprehensive neuroanatomical characterization enhances our understanding of the integral contributions of NTS Phox2b and NTS GABA neurons in modulating complex physiological functions.

Connection pattern of NTS Phox2b and NTS GABA neurons
Previous studies have utilized non-specific axon tracers to explore neural pathways associated with inputs or outputs of the NTS throughout the brain (Ricardo and Koh, 1978;Shioya and Tanaka, 1989;Herbert et al., 1990;Yu and Gordon, 1996;Tanaka et al., 1997;Rinaman, 2010;Ganchrow et al., 2014;Gasparini et al., 2020).Recently, Holt summarized a comprehensive overview of the inputs and outputs of the caudal NTS, revealing widespread inputs from six brain regions (i.e., cortex, subcortical nucleus, diencephalon, mesencephalon, pons, and medulla), along with simultaneous distribution of axonal projections and bidirectional connections among multiple nuclear regions (Holt, 2022).Built upon established neural tracing evidence, our findings provide further insight into inputs and outputs of both excitatory and inhibitory neuronal populations within the NTS, facilitating subsequent investigations aimed at examining function-specific circuits.Specifically, we have demonstrated Photomicrographs of brain regions exhibiting a high density of monosynaptic inputs targeting NTS GABA neurons.A larger number of input neurons innervating NTS GABA neurons was found in many brain regions.Scale bars, 100 μm.
parallel efferent projections of both NTS Phox2b and NTS GABA neurons within the same animals, thereby revealing the spatial distribution patterns of axonal terminals originating from these distinct neuronal populations in their respective target regions.These findings are instrumental in elucidating the circuit mechanisms underlying the differential regulation of homeostasis mediated by both neuronal populations.
We present that NTS Phox2b neurons are recipients of monosynaptic inputs originating from a broad spectrum of brain regions including the medulla oblongata, pons, midbrain, supra-midbrain areas, Quantitative analysis of input neurons innervating the NTS Phox2b neurons and NTS GABA neurons.The relative abundance of input neurons pertinent to NTS Phox2b (A) and NTS GABA (B) neuron populations across distinct brain nuclei was quantified by determining the ratio of tdTomato-tagged afferent neurons within each nucleus relative to the overall neuronal count. 10.3389/fnins.2024.1427384 Frontiers in Neuroscience 10 frontiersin.orgcerebellum and cortex, with a significant density of input neurons located in the IRt, AP, CeA and PVN.Moreover, these NTS Phox2b neurons project their axons towards the medulla, pons, midbrain, and supra-midbrain regions, exhibiting dense outputs particularly in the IRt, Amb, RTN, LPBN, VLPAG and BNST, while conspicuously lacking detectable projections to the cerebellum and cortex.Intriguingly, there exists an array of reciprocal connections between NTS Phox2b neurons and several brain regions, predominantly including the IRt, Amb, RTN, DMNV, BNST and PSTh.Likewise, NTS GABA neurons receive and dispatch monosynaptic inputs and axonal projections to the same array of brain regions as NTS Phox2b neurons, with an increased prevalence of input neurons in the IRt, Gi, AP, CeA, PSTh and PVN, and more pronounced outputs in the IRt, 12 N, LPBN and VLPAG.Additionally, NTS GABA neurons also establish reciprocal connections with the IRt, DMNV, AP and LPBN, illustrating complex patterns of intra-and inter-regional neural circuitries.
The quantitative assessment of synaptic inputs reveals that both NTS Phox2b and NTS GABA neurons receive robust monosynaptic inputs predominantly from regions located in the supra-midbrain areas such as the CeA, PVN, PSTh, and an array of medullary regions including the IRt, AP, parvicellular reticular nucleus (PCRt) and LPGi.Notably, a greater density of midbrain neurons appears to monosynaptically innervate NTS GABA neurons as compared to their NTS Phox2b counterparts.Furthermore, a rigorous exploration of the axonal outputs of these neuronal populations indicates that NTS Phox2b neurons preferentially extend axons towards supra-midbrain nuclei like the BNST and PSTh.In contrast, the projections of NTS GABA neurons are more extensively directed towards pontine regions including the Pr5DM and Pr5VL.This divergent connectivity pattern underscores the roles of NTS Phox2b and NTS GABA neurons in orchestrating functionspecific neural circuits, which may influence various physiological processes or behaviors through distinct neural pathways.
Although we have elucidated the connectivity between the brain and NTS neurons, the present data did not encompass NTS projections to or from the spinal cord.Specifically, the descending projection to the spinal cord is derived exclusively from a small population of neurons located in the ventrolateral NTS (Saper and Stornetta, 2014).The spinal targets of this pathway include respiratory motor neurons as well as the sympathetic preganglionic column (Loewy and Burton, 1978;Dobbins and Feldman, 1994).Additionally, the NTS also receives afferents from the superficial layers of the spinal and trigeminal dorsal horns (Menetrey and Basbaum, 1987).Many of the dorsal horn neurons contributing to this pathway have neurokinin-1 receptors (Al-Khater and Todd, 2009), are activated by visceral stimuli and contain glutamate (Gamboa-Esteves et al., 2001).In conjunction with similarities and distinctions in long-range connectivity, our observations also underline reciprocal innervation between NTS Phox2b and NTS GABA neurons, thereby establishing local circuits of interaction among these neuronal populations.It is noteworthy that while neurons within the NTS receive substantial afferent inputs of visceral origin, the focus of the current study is primarily centered on elucidating the central connective architecture, eschewing considerations related to peripheral inputs.This delineation allows for a more targeted analysis of intracerebral neural pathways, which contributes to a deeper understanding of the regulatory mechanisms underpinning these complex neuronal interactions.

Reciprocal connections of NTS with breathing-related regions in brainstem
The NTS plays an essential role in homeostatic control of breathing.In this study, we elucidate that some brainstem regions establish direct projections to both NTS Phox2b and NTS GABA neurons, forming recurrent circuits that modulate respiratory homeostasis.NTS neurons are integral in processing afferent signals originating from carotid body chemoreceptors, thus playing a crucial role in the modulation of peripheral respiratory chemoreflex pathways.Additionally, NTS Phox2b neurons function as central respiratory chemoreceptors to provide excitatory drive to the respiratory central pattern generator (rCPG) (Fu et al., 2017(Fu et al., , 2019)).Our findings indicate that NTS Phox2b neurons exhibit reciprocal projections with the RTN and LC, both acknowledged as central respiratory chemoreceptors (Wang et al., 2013a,b;Liu et al., 2021).This arrangement suggests the formation of a regulatory network of central respiratory chemoreceptors, instrumental in maintaining respiratory homeostasis.Moreover, NTS Phox2b neurons establish reciprocal projection with the preBötC, BötC, LPBN and K-F nucleus, all of which contribute to rhymogenesis and pattern generation (Del Negro et al., 2018;Krohn et al., 2023).Likewise, NTS GABA neurons also have bidirectional connections with putative central respiratory chemoreceptors and the rCPG as depicted above.To date, experimental data are lacked regarding control of breathing by NTS GABA neurons.Takakura et al. have made initial observations indicating that a subset of NTS GABA neurons interfaces with RTN chemoreceptors, mediating their inhibition in response to lung inflation, thus hinting at the complex regulatory roles these neurons may play within the respiratory control network (Takakura et al., 2007).In addition to the RTN region, pump cells of the NTS, which were first described in 2004 (Ezure and Tanaka, 2004), also projected their axons to the ipsilateral ventrolateral medulla.And axonal arborizations were found in respiration-related areas and their vicinity between the level of the retrofacial nucleus and the level a few millimeters caudal to the obex (Ezure and Tanaka, 1996).
Based on neuroanatomical evidence, activation of NTS Phox2b neurons through its own sensing mechanism or synaptic inputs may provide excitatory drive to breathing, whereas stimulation of NTS GABA neurons is prone to exert a brake-like effect on breathing likely through acting on central chemoreceptors or the rCPG.Of note, both types of population receive monosynaptic input arising from the preBötC, a kernel structure responsible for inspiratory rhythmogenesis.Recent findings demonstrate that in addition to inspiratory motor output, Cdh9/Dbx1-residing preBötC neurons contribute to regulating the balance between calm and arousal behaviors (Yackle et al., 2017).
According to the present and previous data (Yang and Feldman, 2018;Yang et al., 2020), whether SST-expressing preBötC neurons regulate breathing pattern through a feedback circuit mechanism, for example, acting on NTS Phox2b and NTS GABA neurons remains unclear.
The PAG contributes to integrating motor, limbic, and sensory information to modulate behavior-related breathing, such as gasping and vocalization (Subramanian and Holstege, 2010).The stimulation of dorsal PAG and LPAG induces tachypnea and they are mostly involved in active coping strategies, such as fighting or fleeing, while the stimulation of dorsomedial PAG and VLPAG elicited bradypnea, profound respiration, dyspnea, and inspiratory apneas that it is associated with passive coping strategies, such as freezing (Gonzalez-Garcia et al., 2024).The reciprocal projections between NTS Phox2b and NTS GABA neurons and the PAG may reveal circuit mechanisms underlying the above respiratory effects.

Supra-midbrain functional input and output of NTS Phox2b and NTS GABA neurons
Descending inputs from the forebrain provide information regarding emotional, cognitive, and physiological state to the NTS.Thereby, the NTS neurons may orchestrate these behaviors and trigger different autonomic output, including breathing.Our findings demonstrate that the cortex project directly to both NTS Phox2b and NTS GABA neurons, with the putative purpose of promoting behaviors that need to be timed with certain autonomic function such as the breathing cycle, e.g., breath-hold, chewing, swallowing and vocalization.Quantitative analysis of efferent projection from NTS Phox2b and NTS GABA neurons.To quantify the whole-brain efferent projections of NTS Phox2b (A) and NTS GABA (B) neurons, the proportion of axonal projections was calculated as the ratio of the average number of varicosities to the total number of whole-brain projection varicosities.Shao et al. 10.3389/fnins.2024.1427384Frontiers in Neuroscience 13 frontiersin.org In the current investigation, the CeA, PVN and PSTh project heavily to the NTS, in line with previous studies obtained using neural tracing dye (Gasparini et al., 2020;Kim et al., 2022;Huo et al., 2024).Previous studies applied the conventional anterograde tracing method to demonstrate the axonal connections of the NTS with the PVN (Shi et al., 2021;Liu et al., 2023), BNST (Shi et al., 2021), CeA (He et al., 2022) and Arc (Martinez de Morentin et al., 2024), a framework that was further extended using specific cell types of the NTS in the present study.The CeA is the critical centers for processing emotional behaviors, learning and fear response.The amygdala, receiving respiratory inputs, exhibits rhythmic activity that correlates with inspiratory activity signaled by the phrenic nerve root (Onimaru and Homma, 2007), suggesting that the amygdala's spontaneous oscillatory behavior is associated with respiratory functions.In humans, electrical stimulation of the amygdala leads to an apnea (Dlouhy et al., 2015).Therefore, it appears that the CeA-NTS circuits mediate behavioral/ emotional regulation of autonomic output.
The PVN plays imperative roles in the regulation of energy balance and various endocrinological activities, as well as ventilatory homeostasis (Fukushi et al., 2019).It has been shown that electrical stimulation of the PVN in anesthetized rabbits produced an increase in respiratory frequency (Duan et al., 1997); injection of glutamate into the PVN increased electromyographic activity of the diaphragm in anesthetized rats (Yeh et al., 1997).Moreover, disinhibition of the PVN with GABAA receptor antagonist increased both respiratory frequency and tidal volume in conscious rats (Schlenker et al., 2001).The most prominent role of the PVN is its involvement in the mediation of the respiratory response to hypoxia (Ruyle et al., 2019).Furthermore, the role of the PVN in mediating chemoreflex may be specific to hypoxic but not to hypercapnic stimulation (Reddy et al., 2005).However, the neural circuit mechanism through which the PVN regulates ventilation remain to be fully elucidated.The present neural tracing data may provide putative circuit mechanism underlying these effects.

Limitations of the study
In the present investigation, we have elucidated the whole-brain mapping of projections of both NTS Phox2b and NTS GABA neurons.However, the study has certain limitations due to its aim and technical challenges.First, it should be noted that this study did not examine projections to or from the spinal cord, nor did it address potential peripheral inputs to these neuronal populations.Such inputs may originate from critical regions like the carotid body and carotid sinus, as well as relay cardiopulmonary and gastrointestinal information.It stands to reason that NTS Phox2b and NTS GABA neurons are poised to concurrently receive both central and peripheral inputs, thereafter integrating these signals and projecting outputs to specific target neurons.Second, based on injection sites and reporter expression, NTS Phox2b and NTS GABA neurons were primarily distributed in the intermediate and caudal parts of the NTS (bregma −7.0 to −8.0 mm).These neurons did not extensively cover the rostral gustatory portions or the caudal commissural portion of the NTS.Third, the current study did not employ cell type-specific markers to identify target brain regions, such as the use of tyrosine hydroxylase as a marker for locus coeruleus (LC) neurons.This absence of cell type-specific markers could potentially limit the scope of quantitative analyses, particularly in the accurate enumeration of cells and axonal varicosities.

Conclusion
Collectively, we established precise anatomical connections between the specific NTS subpopulations and many brain nuclei.The whole brain mapping of inputs and outputs of both excitatory and inhibitory NTS neurons provides a foundational anatomical framework for forthcoming investigations into the inter-regional pathways involved in the respiratory functions of the NTS.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

FIGURE 1
FIGURE 1 Experimental strategy to target NTS Phox2b and NTS GABA Neurons.(A) Schematic of viral vectors for RV-based trans-synaptic retrograde tracing, including helper viruses with Cre-dependent expression of TVA receptor (AAV-EF1α-DIO-EGFP-T2A-TVA) and N2cG (AAV-EF1α-DIO-N2cG).The RV was genetically modified by pseudotyping with EnvA (CVS-EnvA-∆G-tdTomato). (B) Schematic of viral injection strategy and experimental procedure performed in Phox2b-Cre and Vgat-Cre mice.(C-H) Photomicrograph showing EGFP-expressing NTS Phox2b (left) and NTS GABA (right) neurons (green), tdTomato-expressing input neurons (red) and starter neurons (yellow).In each panel, the bottom images are the enlarged view of the yellow boxed region in the top images.Bottom images (from left to right): EGFP, tdTomato, DAPI and composite merge.Scale bars: 100 μm (top images in each panel), 20 μm (bottom images in bottom panel).

FIGURE 4
FIGURE 4Photomicrographs of brain regions displaying dense monosynaptic inputs to NTS Phox2b neurons.A larger number of input neurons targeting NTS Phox2b neurons was observed in many brain regions.Scale bars, 100 μm.